23, Issue of December. 10, pp. X157-7662,. 1972. Printed in U.S.A.. Partial Resolution of the Enzymes Catalyzing. Photophosphorylation. XII. PURIFICATION.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 24i, No. 23, Issue of December 10, pp. X157-7662, Printed
in
Partial Resolution Photophosphorylation XII.
1972
U.S.A.
of the Enzymes
PURIFICATION AND PROPERTIES COUPLING FACTOR 1*
OF AK
Catalyzing
INHIBITOR
ISOLATED
FROM
CHLOROPLAST
(Received for pubIication, NATHAN
NELSON,$
From the Section
HANNAH
XELSON,$
AND
EFRAIM
RACKER
of Biochemistry and Molecular Biology, Cornell University, Ithaca, New York
SUMMARY 1. Coupling factor 1 from spinach chloroplast dissolved in 7 M urea strongly inhibited the ATPase activity of the coupling factor. The inhibitor was identified with the smallest of the five subunits of the coupling factor. 2. The inhibitor was isolated as a pure protein and was estim.ated to have a molecular weight of 13,000. The amino acid com.position was different from that of the inhibitor of mitochondrial ATPase. Mitochondrial inhibitor inhibited only mitochondrial ATPase; chloroplast inhibitor inhibited only chloroplast ATPase. 3. The chloroplast inhibitor is much more insoluble than the mitochondrial inhibitor and can be kept in solution only in the presence of urea or detergents. Like the mitochondrial inhibitor the chloroplast inhibitor is stable to heat but very sensitive to trypsin. 4. The greater hydrophobicity and affinity of the chloroplast inhibitor to the coupling factor helps to explain some of the physiological differences of energy transduction in mitochondria and chloroplasts and is responsible for the failure of previous attempts to isolate it.
The chloroplast coupling factor 1 (CF]) and mitochondrial coupling factor 1 (Fl) resemble each other with respect to the subunit structure as shown on SDS’ gel electrophoresis (l-3), cold lability (4), function in ATP formation (4, 5), and the ATPase activity after heat acpresence of a Mg ++-dependent tivation (6). However, in one respect the two proteins behave differently when bound to the membrane. The ATP-dependent reversal of energy-linked functions (e.g. “Pi-ATP exchange or * This work was supported by Grant GB-30850X from the National Science Foundation. 3 Present address, Department of Biology, Technion, Haifa, Israel. 1 The abbreviations used are: SDS, sodium dodecyl sulfate; FL, mitochondrial ATPase coupling factor 1; CF,, chloroplast coupling factor 1; DTT, dithiothreitol; Tricine, tris(hydroxymethyl)trypsin treated xvit,h L-l-tosylmethylglycine; TPCK-trypsin, amido-2-phenylethylchloromethyl ketone, a specific chymotrypsin inhibitor, PiUS, A-methylphenazonium methosulfate.
July 26, 1972)
I4850
ATP-dependent reversal of electron transport) which require coupling factors are readily shown in mitochondria but not in chloroplasts under standard conditions of photophosphorylation (7, 8). Pullman and Monroy (9) isolated a mitochondrial protein which specifically inhibited the ATPase activity of purified F1. This F1 inhibitor was proposed to have a key role in the regulation of ATPase activity of F1 in intact mitochondria (9, 10). Until now such an inhibitor could not be detected in chloroplast preparations. An attempt b\- Farron (11) to isolate a CFI inhibitor from the purified coupling factor was unsuccessful. Moreover, no differences were detected between the native CF1 which had no ATPase activity, and the heat-activated CFI which catalyzed ATP hydrolysis. It is the purpose of this communication to describe a method for the isolation of a CF1 subunit which specifically inhibits the ATPase activity of purified CF,. The similarities and dissimilarities of CFI inhibitor with F1 inhibitor, and possible role of the inhibitor in the over-all mechanism of photophosphorylation will be discussed. EXPERIMEKTAL
PROCEDURE
Jfuterials-DEAE-cellulose (from Brown Co.) was washed several times with 0.5 M NaCl, then sequentially with 1 M NaOH, HzO, 1 in HCl, HzO, and finally equilibrated with 20 mM Tris-Cl (pH 8). Digitonin, DTT, ATP, Tricine, Tris, sodium ascorbate, and crystalline bovine serum albumin were obtained from Sigma; acrylamide, methylenebisacrylamide, and SDS from Bio-Rad; TPCK-trypsin and soybean trypsin inhibitor from Worthington; and ammonia-free urea from Schwarz-Mann. Triton X-100 was obtained from Sigma. Analytical Jfethocls-Protein concentration was measured by the method of Lowry et al. (12). ATPase activity was followed by the liberation of “Pi from [y-““P]ATP as previously described (6). SDS gel electrophoresis was performed according to the method of Weber and Osborn (13) with specifications described previously (14). Amino acid analysis was carried out in a Spinco model 120C amino acid analyzer according to the procedure of Spackman eb al. (15). Half-cystine was determined by performic acid oxidation according to the method of Moore (16). Preparations-Spinach chloroplasts (17), F,, and F1 inhibitor (10) were prepared as described in the references. CFI was
7657
7658 prepared by the method of Lien and Racker (18) and only fractions with a fluorescence ratio at A 300:A350 greater than one were used. Heat activation of CFI was performed as described previously (6). RESULTS
We considered the possibility that CFI inhibitor exists, but is tightly bound to the CR molecule. Since urea at high concentrations dissociates CF1 into subunits (19), we searched for the inhibitor in such preparations. When dissolved in 7 M urea CF1 lost coupling activity as well as potential ATPase activity. When such urea-treated preparations were added to heat-activated CFI the ATPase activity of the latter was strongly inhibited (Table I). Urea alone at low concentration had no effect on the ATPase activity. Moreover, CF1 activated by trypsin lost its capacity to inhibit ATPase activity indicating a high sensitivity of the inhibitor to proteolysis. The specificity of the ATPase inhibitor was tested byaddingF1 treated with 7 M urea to activated CFI. As can be seen from Table II, this preparation did not inhibit the ATPase activity of activated I
TABLE
Inhibition
of
Ca++- and
Mg++ATPase
by
urea-treated
CF1
CF1, 2 mg, was dissolved in 1 ml of 7 M urea. The reaction mixture for the Ca++-ATPase assay contained, in a final volume of 1 ml: 30 pmoles of Tricine (pH 8), 4 pmoles of ATP containing about 50,000 cpm of [32P]ATP, 8 pmoles of CaCL, and 2 fig of heatThe react’ion mixture for the Mg++ATPase asactivated CF1. say was the same except that CaClz was replaced by 2 pmoles of MgCl*, and 60 pmoles of sodium maleate (pH 8) were included. Treat.ment with trypsin prior to exposure to urea was performed by incubating 2 mg of CF1 with 50 pg of TCPK-trypsin for 30 min at room temperature in a final volume of 0.5 ml. Solid urea was added to yield a 7 M solution in a final volume of 1 ml. Reaction
mixture
Mg++ATPase
Ca++-ATPase
f~moles P&g
Control. . +50plof7Murea
._........_.....
$ 5 ~1 of CF1 in 7 $
M
urea.
10 ~1 of CFI
in 7 M urea.. in 7 M urea.. in 7 M urea.. + 20 pl of trgpsin-treated CF1 M urea.............
+ 20 ~1 of CFI + 50 pl of CFI
of inhibition
of CFI urea-treated
39.8
5.9
40.3 21.2
3.3
10.5
2.5
6.9 3.2
1.5 in 7
TABLE
Speci$city
prolein/min
14.9 15.0
43.8 II
and F1 Mg++ATPase CF1 and FI
activities
by
Both CF, or F1 were dissolved in 7 M urea at a protein eoncentration of 2 mg per ml. The reaction mixture for the Mg++ATPase assay of CF, was essentially as described in Table I. The reaction mixture for the Mg++ATPase assay of F1 contained, in final volume of 1 ml: 30 pmoles of Tricine (pH 8), 4 rmoles of ATP, 8 pmoles of MgCL, and 0.34 rg of FL. Reaction
mixture
I
Mg++ATPase
(pH 8)
pmolesPdmg fmtein/min Control
CFI. ........................ + 20 jd of CF1 in 7 M urea. ......... + 20 ~1 of Fl in 7 M urea ............ Control Ft ............................ +20alofCF1in7Murea.. ........
+ 20 ~1 of F1 in 7
M
urea ............
11.3
2.0 12.8 74.3 86.7 72.0
CF,. Since purified preparations of F1 have low amounts of F1 inhibitor (lo), purified F1 inhibitor was tested instead. It was ineffective with CFI. Similarly, CFI inhibitor did not inhibit the ATPasc activity of F,. These observations suggest that we are dealing with a specific, rather than with a nonspecific, hgdrophobic interaction between CF, inhibitor and CFI. We have observed2 that pyridine treatment of CF, separated the protein into high and low molecular weight subunits. It was found that the low molecular weight fraction inhibited the ATPase activity of CFI. This observation suggested a very effective procedure for the purification of CFI inhibitor. Purification of CF1 Inhibitor to Homogeneity-About 40 mg of purified CF1 were dissolved in 20 ml of a medium containing 10 mM Tricine, pH 8, and 1 mM EDTA. Twenty milliliters of pyridine were added (with vigorous stirring) and the clear solution was allowed to stand for 10 min at room temperature. Sixty milliliters of distilled water and 0.1 ml of saturated (NH&S04 were then added and the solution, which became turbid, was kept at 4” for 30 min and centrifuged at 10,000 x g for 10 min. The pellet was saved for preparations of CX,p, and y subunits of CF12 and an equal volume of ethanol was added to the supernatant. The preparation was kept overnight at 4” and then centrifuged at 10,000 x g for 10 min. The pellet was freed of excess pyridine by a stream of nitrogen gas and then dissolved in 5 ml of 20 mM Tris-Cl (pH 8) and 7 M urea. The mixture was put on a DEAEcellulose column (1.5 x 4 cm), and elution was carried out with 10 ml of the Tris-urea buffer followed by 10 ml of the same buffer containing 20 mM NaCl. Fractions of 1 ml were collected and tested for inhibition of the Ca ++-ATPase activity of heat-activated CFI . The purity of the fractions was followed by SDS gel electrophoresis. At least 50% of the original activity was recovered in fractions which gave a single band on SDS gel electrophoresis (Fig. 1). Table III summarizes the purification procedure for CF, inhibitor. Procedure for Preparation sf F1 Inhibitor-In view of the efficiency of pyridine in resolving CF1, a similar procedure was developed for the purification of FL inhibitor. Fifty milliliters of water and 100 ml pyridine were added, with vigorous stirring, to 50 ml of twice washed light layer mitochondria in 0.25 M sucrose (50 mg of protein per ml). After 10 min at room temperature, 300 ml of water and 10 ml of saturated ammonium sulfate were added with stirring. The turbid suspension was centrifuged at 20,000 x g for 10 min and the precipitate which contained most of the original proteins was discarded. Five hundred milliliters of ethanol were added to the clear supernatant, and the solution was incubated overnight at 4”. The precipitate which was formed was collected by centrifugation (20,000 X g for 10 min), excess pyridine was removed with a stream of nitrogen gas and the pellet was homogenized with 10 ml of 0.25 M sucrose containing 10 mM Tris-Cl, pH 8.0. After centrifugation and removal of insoluble material, 2.9 g of ammonium sulfate were added to each 10 ml of the supernatant. After 25 min of stirring at 4” the resultant precipitate was removed by centrifugation for 10 min at 14,000 x g and the supernatant was filtered through glass wool to remove floating material. To each 10 ml of the filtrate, 2.25 g of ammonium sulfate were added, the mixture was centrifuged as described above, and the precipitate was dissolved in 10 ml of 0.25 M sucrose. Precipitation with trichloroacetic acid and alcohol was performed as described previously (10). This procedure yielded pure F1 inhibitor with a 3- to 5-fold higher 2 N. Nelson, 11. W. Deters, lished observations.
H. Nelson,
and
E. Racker,
unpub-
7659 yield than obtained by alkaline extraction of mitochondria. With either procedure a small contaminant was occasionally detected which was identified as bovine serum albumin. It was readily removed from the inhibitor by a DEAE-cellulose column as described for the preparation of CFI inhibitor.
stored at -70” in Tris-urea buffer for severalweekswithout loss of activity. It can be seenfrom SDS gelsof CF1 and CF, inhibitor (Fig. 2) that the CR inhibitor correspondsto the lowest molecular weight band seen in purified preparations of CF,.
Properties of CFI Inhibitor-The purified CF, inhibitor is insoluble in water but soluble in the Tris-urea buffer. It was
whenthe two proteins were applied together a separationof the two bandswasobserved. The minimummolecularweight of the CFI inhibitor wasmeasured by SDS-gelelectrophoresis(Fig. 3) with hemoglobin(molecular weight 15,500), ribonuclease (molecular weight 13,700), cytochrome c (molecular weight 12,400), and the large peptide of cytochrome c resulting from cyanogen bromide cleavage (molecularweight 7,650)as markers(20). From thesemeasurements,the molecularweight of CFI inhibitor appearsto be 13,000 and that of F, inhibitor 9,500. The latter value is slightly lower than that (10,500)obtained by Brooks and Senior (2). Table IV showsthe amino acid compositionof CF1 inhibitor. The protein is free of tyrosine and contains 1 residue each of histidine, methionine, cystine, and phenylalanineper mole, with a minimummolecularweight of 13,000. An aminoacid analysis of F1 inhibitor confirmed the finding of Brooks and Senior (2) that it is lacking threonine, proline, and methionine. Threonine and proline were relatively high in the CF, inhibitor and the presenceof a methionine residue was confirmed by cyanogen bromide cleavage. In contrast to 6 histidine residuesfound in
This location
the F, inhibitor,
on SDS gels is similar
only 1 histidine
to that of F1 inhibitor,
but
residue was present in the CFI
inhibitor. As documentedin Fig. 4, about 1.1 pg of CFI inhibitor gave 50% inhibition of Ca++-ATPaseactivity of 1.5 pg of heat-activated CFI. In the presenceof 0.5% Triton, the Ca++-ATPase activity
FIG. 1 (left). The SDS gel electrophoresis pattern of CFI inhibitor. The CFlinhibitor (30 rg) was incubated for 2 hours at room temperature in a medium containing 7 M urea, 1% SDS, and 1% mercaptoethanol. Electrophoresis in SDS polyacrylamide gel was carried out (13, 14) for 5 hours at 7.0 ma per tube. The gel was stained with Coomassie brilliant blue and after destaining was scanned at 600 nm in an attachment of the Gilford spectrophotometer. FIG. 2 (right). The SDS gel electrophoresis pattern of purified CF1 and CF1 inhibitor. Experimental conditions were as described in the legend of Fig. 1 except that 60 pg of CFI and 10 pg of CF1 inhibitor were used.
was slightly
stimulated
and the sensitivity
toward
CFI
inhibitor wasincreasedabout 2-fold. This increasein sensitivity by Triton might be causedby a prevention of nonspecificbinding of Cf inhibitor to CF,, an assumptionsupportedby the data presentedin Table V. Inclusion of 0.5% Triton after heat activation facilitated
the loss of ATPase
activity by storage overnight.
Dilution of the activated enzyme with 1 mM EDTA and 10 mM
Cyt.
C. (CNBr)
F, inhibitor \
III procedure for CFI inhibitor TABLE
Purification
The reaction mixture for the Ca++-ATPase assay was as described in the legend of Table I except that 1.5 pg of heat-activated The specified proCF1 were used and 0.5’% Triton was included. tein fractions were added in 7 M urea solution. Yield
FW3iOlI
I
I
Amount required for 50% inhibition
I
I
3.9
4
I
IOQ
1. CF,. . . . . . . .. . .. 2. Pellet after extraction with 20% pyridine.................,.........,..... 3. Pellet after precipitation with 50% ethanol . . . . .. 4. Combined fractions from DEAE-column which gave single band on SDS gels.
11 50 2.5 0.6
4.1 t’f!w
I
4.2
FIG. 3. Determination of the molecular weight of CF1 inhibitor and F1 inhibitor by gel electrophoresis in the presence of SDS. The experiment was performed with 15 pg of the proteins as described in the legend of Fig. 1 except that the amount of methylenebisacrylamide was doubled to obtained better resolution. Cyt. c. (CNBr), the large peptide resulting from cyanogen bromide cleavage of cytochrome c; R. N., ribonuclease; H. H., human hemoglobin.
TABLE Amino
acid
composition
T
Amino acid
IV
TABLE of CFI
Inhibition
inhibitor
inhibiior Amount per 100 ~moles of amino acid
Residues per molecule (ml wt 13,000)
pmoles Lysine ....................... Histidine ..................... Ammonia ..................... Arginine ..................... Cysteic acid. ................. Aspartic acid. ................ Threonine ................... Serine ........................ Glutamic acid. ............... Proline ....................... Glycine ....................... Alanine ....................... Valine ........................ Methionine ................... Isoleucine.................... Leucine...................... Tyrosine ..................... Phenylalanine................
Ca++-ATPase
of
4.18 0.53 20.55 9.99 0.83 13.36 7.48 5.67 12.69 3.14 6.94 7.14 5.03 0.84 9.94 11.29 0 0.95
5 1 23 11 1 15 9 6 15 4 8 8 6 1 11 13 0 1
V
activity by low concentration during prolonged storage
of CFI
The CF1 (1.53 mg per ml) was activated by heating for 4 min at 64” in the absence of digitonin. After activation, Triton was added in a final concentration of 0.5%. After 28 hours of incubation at room temperature the enzyme was assayed for Ca+&ATPase activity. The conditions of the assay were described in Table I except 3 pg of activated CF, were used. Where indicated, 25 pg of TPCK-trypsin were included in the reaction mixture. The activit,y of the control before incubation was 22.8 rmoles of Pj per mg of protein per min. Ca++-ATPase Reaction mixture Control
+ Trypsin
flmoles PJmg
1. 153~gofCF1in1ml........__...... 2. 153~gofCF1in0.1ml.............. 3. 153 pg of CF1 in 0.1 ml + 0.7pg of CFi inhibitor..................... 4. 153 pg of CFi in 0.1 ml + 1.75 fig of CFiinhibitor....................
TABLE Effect
of TPCK-trypsin
The assay mixture for with 1.5 pg of heat-treated ~1) in 7 M urea was added trypsin as indicated.
protein/w&
17.8 9.4
23.6 18.3
8.5
19.2
6.7
15.3
VI
and
heat on CFI
Ca++-ATPase activity CF1 was used. The to t.he assay mixt,ure
inhibitor (see Table I) CFi inhibitor (5 with or without
Addition to assay mixture
Ca++-ATPase pmoles Pi/mg
CFi (heat-t,reated). CF1 + 1.75 pg of CFi inhibitor CF, + 5 rg of TPCK-trypsin.. CFi + 1.75 pg of CFi inhibitor trypsin CF1 + 1.75 pg of CF1 inhibitor for 5 min in 100”
in 7 M urea.. +
GF,
inhibitor
Protective
ATPase of CFI by FIG. 4. Inhibition of the Ca ++-dependent purified CF1 inhibitor. The reaction mixture contained, in a final volume of 1 ml: 30 pmoles of Tricine (pH S), 4 pmoles of ATP, 8 pmoles of CaC12, 0.5oj, Triton where indicated, and 1.5 fig of heat-activated CFI (6).
pH 8, diminished this effect, as did inclusion of trypsin in the reaction mixture. This suggests that the inhibition was caused by the CFI inhibitor which was previously removed by heat and was reattached to the CF! during the overnight incubation Table VI shows the effects of temperature and trypsin on the CFI inhibitor. -4s can be seen, the inhibitor is heat-stable but very sensitive to trypsin. addition of small amounts of trypsin to the assay mixture completely abolished the inhibition, even when an excess of inhibitor was added to the mixture. In order to explore how the CFi inhibitor interacted with CS, advantage was taken of the availability of specific antisera against the individual subunits of CFi .2 The experiment shown in Table VII was made possible by our observation that none of
32.6 3.7
antisera
against
the
individual
subunits,
when
added
singly,
of antibody
VII
to y subunit by CF1 inhibitor
of CFI
against
inhibition
The reaction mixture for Ca ++-ATPase was as described in the legend of Table I with 1.5 rg of heat-activated CFI. Antiserum or control serum (10 ~1) w-as added to the reaction mixture as indicated, in the presence and absence of 1.5 rg of CFI inhibitor. Cat+-ATPase activity
Trick,
the
effect
TPCK-
incubated
TABLE pg
prolein/min
21.7 3.9 30.4
Additions -CFI
inhibitor pm&s
None......................... Control serum................ Ant&........................ Anti-fi........................ Anti-y. Anti-a.
+CFI
inhibitor
Pi/mg protein/min
36.4 32.2 36.1 38.8 37.4 34.7
16.7 10.1 15.1 11.5 33.8 14.5
inhibited the 4TPase activity of CFi.2 It can be seen from the data that only the antiserum against the y subunit of CF1 effectively
The
interfered
added
CFI
with
the
inhibitor
inhibitory
did
effect
not
of CFi
interfere
inhibitor.
with
either
7661 PMS-mediated cyclic photopl~ospl~orylation nor any of the other reactions of isolated chloroplasts in which CF1 is involved. Preparafion~jCF~ Freeoj 6 Swbunitsand CFl Inhibitor-It was shown previously (21) that when the ATPase of CFI was activated by heat, coupling activity was lost. We have subsequently observed (6) that when the heat treatment was performed in the presence of digitonin, both ATPase and coupling activit,y were preserved. When such an activated preparation of CFI was passed through a Sephadex G-200 column, which was equilibrated with buffer containing digitonin, two minor bands observed in SDS gels were no longer detected (Fig. 5). Yet both the ATPase and the coupling activity were unaffected by this fractionation procedure. DISCUSSION
The similarity between chloroplasts and submitochondrial particles was discussed in detail elsewhere (1, 22). One of the main differences is that chloroplasts, as well as subchloroplast particles, exhibit virtually no ATPase activity and catalyze no 32Pi-ATP exchange under conditions that permit photophosphorylation to occur. It, therefore appears that in chloroplasts one of the steps in the transphosphorylation of ADP is not readily reversed. Pullman and Monroy (9), Horstman and Racker (lo), and Asami et al. (23) suggested that the F, inhibitor modifies Fl in such a manner that reverse reactions with ATP are prevented, while the forward reaction with ADP proceeds. It was also proposed (1) that in chloroplasts a more tightly bound inhibitor of CF1 may be responsible for the lack of reversibility of chloroThe data presented in this paper supplast-catalyzed reactions. port this assumption. It, was shown that the CFi inhibitor is more firmly bound to CF1 and can only be removed by heat treatment in the presence of digitonin followed by fractionation with Sephadex G-200. The need for the detergent can be explained by differences in solubility. Purified F1 inhibitor is soluble in water (9, 10) whereas CF, inhibitor is soluble only in
the presence of urea or detergents. This finding also helps to explain the observation that CF, heated without digitonin lacks coupling activity (21). We propose that the hydrophobic CF1 inhibitor, which is released on heating in the absence of a detergent, recombines with CF, at a site required for binding to the membrane. The hydrophobicity of the CF, inhibitor also esplains the lack of an effect by salts, which were noted to interfere with the binding of F1 inhibitor to FI (9). It may also account for the larger amounts of CFI inhibitor required for inhibition of ATPase activity, assuming nonspecific interaction with other hydrophobic regions of CFI. However, in the preseuce of 0.5% Triton and concentrated CFI, the endogenous CFI inhibitor was enough to give 50% inhibition after overnight storage. Purified CF, inhibitor added to the incubation medium in concentrations lower than the concentration of CF1 gave further inhibition (Table V). The removal of CF1 inhibitor by heat treatment (Fig. 5) and its sensitivity toward trypsin (Table I), suggests that both treatments activate the latent ATPase of CFI either by destroying the inhibitor or by removing it from the active site of the enzyme. On the other hand, DTT which activates the ATPase (8) does not remove the inhibitor, since after removal of DTT the ATPase activity reverts to its latency (21). It therefore appears that DTT somehow interferes with the interaction of the inhibitor with the active site of CFI. In line with this conclusion is the experimental observation that inhibition of heat-activated CF, by added inhibitor is also counteracted by DTT. Further studies on the mechanism of action of CF, inhibitor should be carried out with inhibitor-free CFI and completely resolved subchloroplast particles similar to the mitochondrial ASU particles (submitochondrial particles sonicated in ammonia and exposed sequentially to Sephadex and urea (24)). Unfortunately, such particles are not as yet available. Highly resolved STA particles (subchloroplast particles treated with silicotungstate (22)) might be used, but they show poor recovery of photophosphorylation upon addition of CFi. We are now looking for methods to prepare resolved subchloroplast particles which can be reconstituted with better recovery of photophosphorylation. REFERENCES 1.
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R~CKEI~, NELSOX,
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weight CFI subunits by FIG. 5. Removal of the low molecular heat treatment and Sephadex G-200 column chromatography in the presence of digit.onin. About 5 mg of CFI in a final volume of 1 ml were heated at 64” for 4 min as previously described (6) but The sample was passed through in the presence of 1% digitonin. a Senhadex G-200 column (1.25 X 60 cm) which was equilibrated with’s solution containing 10 rnbr Tricine (pH 8), 1 rn; EDTA, 1 rnM DTT, 1 mM ATP, 200 rnM (NH4)2S04, and 0.2% digitonin. The digitonin was necessary to obtain the separation but sometimes it tended to precipitate and the column was blocked as a result. Fractions of 2 ml were collected and tested for Ca++ATPase, coupling activity, and their pattern on polyacrylamide gels containing SDS elect,rophoresis. A, untreated CFI (60 rg); 13, treated CF1 (50 pg). The conditions for SDS gel electrophoresis were as described in the legend of Fig. 1.
6. NELSOK, N., NELSON, Chem.247,6506-G510 7. AVRON, M., CXISARO, Chem. 240, 1381-1386
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